Having considered localization of respiratory functions in mitochondria and in prokaryotic cells, it’s time to return to the eukaryotic context and follow a molecule of pyruvate across the inner membrane of the mitochondrion to see what fate awaits it inside.
In the presence of oxygen, pyruvate is oxidized fully to carbon dioxide, and the energy released in the process is used to drive ATP synthesis. The first stage in this process is a cyclic pathway that is a central feature of energy metabolism in almost all aerobic chemotrophs. An important intermediate in this cyclic series of reactions is citrate, which has three carboxylic acid groups and is therefore a tricarboxylic acid. For this reason, this pathway is usually called the tricarboxylic acid (TCA) cycle. It is also commonly referred to as the Krebs cycle in honor of Hans Krebs, whose laboratory played a key role in elucidating this metabolic sequence in the 1930s.
The TCA cycle metabolizes acetyl coenzyme A (acetyl CoA), which consists of a two-carbon acetate group linked to a carrier called coenzyme A. (Coenzyme A was discovered by Fritz Lipmann, who shared a Nobel Prize with Krebs in 1953 for their work on aerobic respiration.) Acetyl CoA arises either by oxidative decarboxylation of pyruvate or by the stepwise oxidative breakdown of fatty acids. Regardless of its origin, acetyl CoA transfers its acetate group to a four-carbon acceptor called oxaloacetate, thereby generating citrate. Citrate is then subjected to two successive decarboxylations and several oxidations, leaving a four-carbon compound from which the starting oxaloacetate is regenerated.
Each round of the TCA cycle activity involves the entry of two carbons (as the acetate from acetyl CoA), the release of two carbons as carbon dioxide, and the regeneration of oxaloacetate. Oxidation occurs at five steps: four in the cycle itself and one in the reaction that converts pyruvate to acetyl CoA. In each case, electrons are accepted by coenzyme molecules. The substrates for the TCA cycle are therefore acetyl CoA, oxidized coenzymes, ADP, and Pi, and the products are carbon dioxide, reduced coenzymes, and a molecule of ATP (or GTP, a closely related nucleotide).
With this brief overview in mind, let’s look at the TCA cycle in more detail, focusing on what happens to the carbon molecules that enter as acetyl CoA and how the energy released by each of the oxidations is conserved as reduced coenzymes.
As carbon enters the TCA cycle in the form of acetyl CoA, the glycolytic pathway ends with pyruvate, not acetyl CoA. To get from pyruvate to acetyl CoA requires the activity of pyruvate dehydrogenase (PDH), a huge multiprotein complex that has a molecular weight of about 4.6 x 106 and consists of three different enzymes, five coenzymes, and two regulatory proteins. These components work together to catalyze the oxidative decarboxylation of pyruvate:
The TCA cycle begins with the entry of acetate in the form of acetyl CoA. With each round of the TCA cycle activity, two carbon atoms enter in organic form (as acetate), and two carbon atoms leave in inorganic form (as carbon dioxide). In the first reaction (TCA-1), the two-carbon acetate group of acetyl CoA is added onto the four-carbon compound oxaloacetate to form citrate, a six-carbon molecule. This condensation is driven by the free energy of hydrolysis of the thioester bond and is catalyzed by the enzyme citrate synthase. That citrate is a tricarboxylic acid—the class of compounds giving the TCA cycle its name.
Next, one will notice that four of the eight steps in the TCA cycle are oxidations. This is evident because four steps involve coenzymes that enter in the oxidized form and leave in the reduced form. Each of these reactions is catalyzed by a dehydrogenase that is specific for the particular substrate.
With the regeneration of oxaloacetate, one turn of the cycle is complete. It can summarize what has been accomplished by noting the following properties of the TCA cycle:
Acetate enters the cycle as acetyl CoA and is joined to a four-carbon acceptor molecule to form citrate, a six-carbon compound.
Decarboxylation occurs at two steps in the cycle so that the input of two carbons as acetate is balanced by the loss of two carbons as carbon dioxide.
Oxidation occurs at four steps, with NAD+ as the electron acceptor in three cases and FAD as the electron acceptor in one case.
ATP is generated at one point, with GTP as an intermediate, in animal cells.
One turn of the cycle is completed upon regeneration of oxaloacetate, the original four-carbon acceptor.
By summing the eight component reactions of the TCA cycle, we arrive at an overall reaction (In this and subsequent reactions, protons and water molecules are not explicitly shown if present only for charge or chemical balancing.) This reaction is written as:
Because the cycle must, in effect, occur twice to metabolize both of the acetyl CoA molecules derived from a single molecule of glucose, the summary reaction on a per-glucose basis can be obtained by doubling reaction, then add to this reaction the summary reactions for glycolysis through pyruvate and for the oxidative decarboxylation of pyruvate to acetyl CoA arrive at the following overall reaction for the entire sequence from glucose through the TCA cycle:
Considering this summary reaction, two points may strike- how modest the ATP yield is thus far, and how many coenzyme molecules are reduced during the oxidation of glucose. However, it must also recognize the reduced coenzymes NADH and FADH2 as high-energy compounds in their own right. The transfer of electrons from these coenzymes to oxygen is highly exergonic.
For the release of that energy, it must look to the remaining stages of respiratory metabolism—electron-transported oxidative phosphorylation. Before doing so, however, it will consider several additional features of the TCA cycle: its regulation, its centrality in energy metabolism, and its role in other metabolic pathways.
The author is an associate professor (retd.) and former head of the department of botany at Ananda Mohan College.